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Citation: Zhou, M.; Huo, M.; Wang,

J.; Shi, T.; Li, F.; Zhou, M.; Wang, J.;

Liao, Z. Identification of Tartary

Buckwheat Varieties Suitable for

Forage via Nutrient Value Analysis at

Different Growth Stages. Int. J. Plant

Biol. 2022, 13, 31–43. https://

doi.org/10.3390/ijpb13020005

Academic Editor: Adriano Sofo

Received: 25 February 2022

Accepted: 25 March 2022

Published: 29 March 2022

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Article

Identification of Tartary Buckwheat Varieties Suitable forForage via Nutrient Value Analysis at Different Growth StagesMengjie Zhou 1, Mingxing Huo 1, Jiankang Wang 1, Tiantian Shi 1, Faliang Li 2, Meiliang Zhou 3 ,Junzhen Wang 2,* and Zhiyong Liao 1,*

1 College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, China;[email protected] (M.Z.); [email protected] (M.H.);[email protected] (J.W.); [email protected] (T.S.)

2 Liangshan Prefecture Academy of Agricultural Sciences, Liangshan Prefecture, Xichang 615000, China;[email protected]

3 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China;[email protected]

* Correspondence: [email protected] (J.W.); [email protected] (Z.L.)

Abstract: It is important to select buckwheat varieties suitable for foraging and determining theirbest harvest time as increasing attention was paid to the forage value of buckwheat. Here, eighttartary buckwheat varieties were identified as suitable for forage based on their potential foragevalue through assaying the contents of ash, crude protein, crude fiber, crude fat, acid detergent fiber,neutral detergent fiber, nitrogen free extract, calcium, phosphorus, total flavonoids, and rutin in thesetartary buckwheat varieties at flowering, pustulation, and mature stages, respectively. In addition,analysis of relative feed value (RFV), relative forage quality (RFQ), and principal component analysis(PCA) based on the assayed contents was applied for comprehensive evaluation of these tartarybuckwheat varieties. Results showed that all the eight tartary buckwheat varieties possessed potentialhigh forage value as their RFV is from 121.31% to 217.39% and RFQ from 117.26% to 224.54% at allthree stages. In particular, both RFV and RFQ values of PS-07 reached the highest at the floweringstage among the eight tartary buckwheat varieties, followed by CQ-3 and EWPS. Accordingly, thecomprehensive scoring of principal component values of PS-07 and CQ-3 are relatively higher at theflowering stage. Our research thus revealed that the eight tartary buckwheat varieties are all suitablefor forage, and also provided an experimental basis for selecting the eight tartary buckwheat varietiesharvested at different growth stages for livestock forage.

Keywords: tartary buckwheat; forage; relative feed value; relative forage quality; principal compo-nent analysis

1. Introduction

Buckwheat known as triangular wheat is a dicotyledonous plant of the genus Fagopy-rum which belongs to the family Polygonaceae [1]. Cultivated buckwheat includes twospecies originating from China: common buckwheat (Fagopyrum esculentum) and tartarybuckwheat (Fagopyrum tartaricum), among which tartary buckwheat is extensively culti-vated as an alternative crop in China as early as 1000 BC. Tartary buckwheat is regarded asa popular food and forage crop with strong ecological adaptability due to its well-adapted,short growth period, highly resistant to barren soil and insect pests [2]. As a well-adaptedand climate-resilient crop, tartary buckwheat is very suitable for planting in some remoteareas of China such as Liangshan Yi Autonomous Prefecture in Sichuan Province or Qianxi-nan Buyei and Miao Autonomous Prefecture in Guizhou Province where barren land isnot good for major crop growth, but tartary buckwheat can still grow normally [3]. In themountain area of western China with a long history of tartary buckwheat cultivating, sometartary buckwheat landraces, especially those with amounts of large leaves, are commonly

Int. J. Plant Biol. 2022, 13, 31–43. https://doi.org/10.3390/ijpb13020005 https://www.mdpi.com/journal/ijpb

Int. J. Plant Biol. 2022, 13 32

used as forage. Compared with other forages like oats and maize, tartary buckwheat has ob-vious advantages of production and preservation [4]. At the same time, tartary buckwheathas significant superiorities in view of forage as its nutritional ingredients not only containcommon basic ingredients such as proteins, fats, celluloses and sugars, but are also rich inantioxidant flavonoids such as rutin, quercetin, catechins, and catechins which could satisfyvarious livestock’s demand for feed [5,6]. Due to these advantages, tartary buckwheat isdeemed as a high-quality forage resource with promising development potential.

With the social development and ever-increasing world population, the current percapita consumption of animal husbandry products is also gradually rising, such as milk,milk powder and common edible meat. In addition to the increasing demand for animalhusbandry products, people are paying more attention to the quality of animal husbandryproducts, which largely depend on the quality of forage [7]. Currently, the whole plantsof crops alfalfa [8–10], sorghum [11], and forage maize [12] are the main forage widelyused; however, they have obvious shortcomings such as low protein content and utiliza-tion rate [13], poor environmental adaptability [14], and high soil water consumption forgrowth [15]. In addition, these forages usually lack antibacterial and antiviral bioactivesubstances that could greatly benefit the health of the livestock. Although addition of somespecific substances such as vitamins and antibiotics could confer to forage more compre-hensive function, antibiotics added to livestock feed, after ingestion, were incompletelymetabolized and poorly absorbed in the gastrointestinal tract, resulting in excretion ofparent compounds and metabolites [16], which could bring potential harm to consumer’shealth via livestock products. Therefore, antibiotics are currently rarely used in livestockproduction, which might lead to decreased disease resistance. Extensive research has beendone over the last couple decades to search for natural alternatives to in-feed antibiotics,and some plant compounds have been identified to have great potentials [17]. Tartary buck-wheat, when used as forage, might be full of nutritional value since its component could notonly satisfy the livestocks’ need for basic nutrients, but also improve the livestocks’ abilityto resist disease as it was reported that buckwheat flavonoids displayed anti-oxidation [18],anti-bacteria [19], and anti-virus activities [20]. Studies have indicated that both the wholetartary buckwheat plants and grains can be used as potential high-quality forages. Cui et al.confirmed that tartary buckwheat could alleviate rumen methane emissions by improvingthe morphology of gut microbes [21]. Scuderi et al. reported that adding buckwheatingredients to forage could effectively improve the morphology of intestinal microbes inruminant animals by affecting the fermentation activity of ruminant rumen microbes [22].Amelchanka et al. also found that addition of a proper amount of buckwheat to foragecould improve the palatability and productivity of dairy cows [23]. Therefore, finding andbreeding novel tartary buckwheat varieties suitable for forage might be of great value.

In our previous research aiming to select buckwheat varieties suitable for forage, eighttartary buckwheat varieties with large leaves, tall plants, thick stems, more branches, andhigh production of the whole plant were bred in Liangshan Yi Autonomous Prefectureof Sichuan Province, including Xiqiao No. 1 (XQ-1), Youqiao No. 1 (YQ-1), Meng-09125(M-09125), Chuanqiao No. 3 (CQ-3), Zhaoku No. 1 (ZK-1), Ewu Pedgree selection (EWPS),Buyue (BY), and Pedgree selection-07 (PS-07). In order to further clarify the potentialforage application value of these eight tartary buckwheat varieties, so as to select thetartary buckwheat varieties that are relatively suitable for forage use, especially consideringthat the component content of the whole plant of tartary buckwheat in different growthstages, including some very important flavonoids and other active components changegreatly with the change of growth stage, the main nutrients related to forage use and thecontents of flavonoids with important biological activities in these eight tartary buckwheatvarieties at different growth stages were determined. In addition, a more comprehensiveanalysis and evaluation was carried out using relative forage value (RFV) and relative feedquality (RFQ) to further evaluate the potential forage value of these eight tartary buckwheatvarieties. This study provided a strong theoretical basis for the selection of these eighttartary buckwheat varieties as forage, especially in the harvest stage of forage.

Int. J. Plant Biol. 2022, 13 33

2. Materials and Methods2.1. Plant Material

Whole-crop of eight tartary buckwheat varieties including Xiqiao No. 1 (XQ-1),Youqiao No. 1 (YQ-1), Meng-09125 (M-09125), Chuanqiao No. 3 (CQ-3), Zhaoku No.1 (ZK-1), Ewu Pedgree selection (EWPS), Buyue (BY), and Pedgree selection-07 (PS-07)were from Mengzi feed factory, Zhaojue County, Liangshan Yi Autonomous Prefecture,Sichuan Province, China. The samples of buckwheat varieties were harvested and driedin the whole field, and 1.0 kg of samples were randomly selected for quality testing andanalysis from each field to ensure that the samples were representative.

2.2. Nutritional Analysis2.2.1. Materials Pretreatment

Tartary buckwheat samples were dried at 65 ◦C for 72 h (Shanghai Longyue Co., Ltd.,Shanghai, China), smashed, and then ground through a 0.27 mm screen (Wenzhou DingliMedical Instrument Co., Ltd., Wenzhou, China).

2.2.2. Ash Content

Ash content was determined according to the procedure by Neu et al. [24]. In brief,5.0 g of each tartary buckwheat sample were charred and then incinerated in the mufflefurnace (Shanghai Lichenkeyi Co., Ltd., Shanghai, China) at 550 ◦C for 5.5 h. The ash wascooled at room temperature and weighed.

2.2.3. Crude Protein Content

CP was determined according to the procedure by the Folin–Ciocalteu method. Briefly,0.3 g of each plant sample were digested with 16 mL 1.0 mol/L NaOH at 50 ◦C for 4 h. Theproduct was diluted by 500 times. In addition, 4 mL Folin-Phenol reagent A were added to0.8 mL diluted sample and then placed at 37 ◦C water bath (Hangzhou Bioer TechnologyCo. Ltd., Hangzhou, China) for 10 min, followed by addition of 0.4 mL Folin-Phenol regentB and for another 30 min. Bovine Serum Albumin (Beyotime Biotechnology Co., Ltd.,Shanghai, China) was used as the standard. CP content was determined by the absorbanceat 750 nm using an ultraviolet spectrophotometer (Shanghai Meipuda instrument Co., Ltd.,Shanghai, China).

2.2.4. Crude Fiber Content

CF was determined according to the procedure by D’Heer et al. [25]. Furthermore, 1.0 gof each plant sample was weighed into filter bags. In addition, 150 mL 0.13 ± 0.05 mol/LH2SO4 and 150 mL 0.23 ± 0.05 mol/L KOH of each sample were pre-heated to 95 ◦C inthe reaction vessel, respectively, after submerging the samples, brought to a boiling pointby a heating plate (Shuzhou Guofei Laboratory Instrument Co., Ltd., China) as soon aspossible. Digestion times, 30 min for both steps, were measured from the moment thesamples were put in the vessel. After the first and the second digestion, samples werewashed respectively two and three times for 5 min with boiling water. After expelling theexcess water, the bags were soaked in acetone for 3–5 min. The air-dried filter bags werecompletely dried for 4 h at 103 ◦C (Shanghai Longyue Co., Ltd., China) and ashed for 2 h at550 ◦C. CF-values were corrected for ash content.

2.2.5. Crude Fat Content

EE was determined according to the procedure by Thiex et al. [26]. In addition, 2.0 g ofeach plant sample were dissolved in 10 mL 12 mol/L HCL with water bathed at 70–80 ◦Cfor 50 min (Hangzhou Bioer Technology Co. Ltd., China). Furthermore, 10 mL ethanol and25 mL absolute ether were mixed with the sample by shaking, and then allowed it to standfor 15 min. Water bathed at 100 ◦C (Hangzhou Bioer Technology Co. Ltd., China) to makethe samples be dry. Finally, the samples were completely dried for 2 h at 105 ◦C (ShanghaiLongyue Co., Ltd., China), and then weighed.

Int. J. Plant Biol. 2022, 13 34

2.2.6. Acid or Neutral Detergent Fiber Content

ADF and NDF were determined according to the procedure by D’Heer et al. [25]. Inaddition, 1.0 g of each plant sample were weighed into filter bags, after submerging in 2 Lpreheated acid detergent solution and neutral detergent solution at 95 ◦C by water bath(Hangzhou Bioer Technology Co. Ltd., China), respectively, brought to a boiling point by aheating plate (Shuzhou Guofei Laboratory Instrument Co., Ltd., China) as soon as possible.After digestion for 60 min, bags were washed 3 times for 5 min with boiling water andthen, after removal of the free water, soaked in acetone for 3 min with agitation. Finally, theair-dried filter bags were completely dried for 4 h at 103 ◦C (Shanghai Longyue Co., Ltd.,China) and ashed for 2 h at 550 ◦C (Shanghai Lichenkeyi Co., Ltd., China).

2.2.7. Nitrogen Free Extract Content

The percentage NFE was then calculated by the laboratory using the formula NFE= 100 − (crude protein + crude fat + crude fiber + moisture + ash) % [27], moisture wasdetermined according to the procedure by Chinese standard GB/T 6435-2014. In addition,5.0 g of each plant sample were weighed and completely dried for 4 h at 103 ◦C.

2.2.8. Phosphorus Content

Phosphorus content was determined according to the Chinese standard (GB/T 6437-2018). Furthermore, 5.0 g of each plant sample were processed as 4.2.2, dissolved with10 mL 50% HCL and made it to 100 mL with water. In addition, 1 mL supernatant wasmixed with 10 mL Vanadium ammonium molybdate. Phosphorus content was run throughan ultraviolet spectrophotometer (Shanghai Meipuda Instrument Co., Ltd., China) todetermine the absorbance at 400 nm.

2.2.9. Calcium Content

Calcium content was determined according to the procedure by Chinese standards(GB/T 6436-2018). In addition, 5.0 g of each plant sample were processed as 4.2.2, dissolvedwith 10 mL 25% HCL and few drops of HNO3, boiled and made it to 100 mL with water.Furthermore, 10 mL of sample were added with 50 mL H2O, 10 mL starch solution, 2 mLTriethanolamine (C2H8N2), 1 mL Ethylenedia-mine (EDA, C6H15NO3), and a drop ofMalachite Green (C23H25CN2), added with 3 drops of calconcarboxylic acid; then, Cacontent was assayed by titration with EDTA.

2.2.10. Total Flavonoids Content

Total flavonoids were determined according to the procedure by Bhandari et al. [28].All samples were dissolved in 80% methanol (v/v) with supersonic extraction. Rutin(Beijing Solarbio Science & technology Co., Ltd., Beijing, China) was used as standard. Inaddition, 2.5 mL of each extract (80 mg/mL) were mixed with 5 mL 0.1 mol/L AlCl3 and7.5 mL 1 mol/L CH3COOK into 25 mL volumetric flask after centrifugation, diluted with80% methanol to volume. The absorbance read at 420 nm after 30 min.

2.2.11. Rutin Content

The rutin present in the tartary buckwheat was quantified by HPLC [29]. All sampleswere dissolved in 80% methanol (v/v) with supersonic extraction, and filtered by 0.45 µmmembrane for injection. Rutin (Beijing Solarbio Science & Technology Co., Ltd., Beijing,China) was used as the standard. Eclipse Plus C18 5 µm, 4.6 mm × 250 mm column wasused in this HPLC (LC-20AT, Shimadzu, Shang hai, China.) analysis with a flow rate of0.5 mL/min. The mobile phase consisted of (A) ultrapure water and (B) methanol with thefollowing gradient: 50% B (0.01–18 min), 80% B (18.01–21 min), 50% B (21.01–30 min). Therutin was detected at a wavelength of 350 nm.

Int. J. Plant Biol. 2022, 13 35

2.3. Relative Feed Value and Relative Forage Quality

Dry matter intake, digestible dry matter, total digestible nutrients, relative feed value,and relative feed quality were calculated by formulas [30]:

DMI (%BW) = 120/NDF (%DM), (1)

DDM (%DW) = 88.9 − 0.779 × ADF (% DM), (2)

TDN = 82.38 − (0.751 × ADF), (3)

RFV = DMI (% BW) × DDM (% DM)/1.29, (4)

RFQ = TDN × DMI/1.23. (5)

2.4. Statistical Analyses

Microsoft Excel 2016 was used for data input, processing of the original data, andbasic statistical analysis. IBM SPSS Statistics 23 (SPSS software version 23) was used toperform one way analysis of variance (ANOVA) to assess the difference of forage nutritionalquality of each tartary buckwheat growth period, and principal component analysis (PCA)was used to screen the principal components of tartary buckwheat samples by using thecorrelated nutrient indicators of the samples. Fisher’s Least Significant Difference (LSD)was used to compare the means when the treatment effects were significant at α = 0.05, andthe data are present as the mean ± standard errors of three independent replicates.

3. Results3.1. Nutritional Values of Eight Tartary Buckwheat Varieties

Results in Table 1 showed that the content of ash in eight tartary buckwheat varietiesat the flowering stage varied between 14.54% and 17.49%, the content of crude protein (CP)between 3.15% and 7.08%, the content of fiber (CF) between 6.00% and 13.50%, the contentof crude fat (ether extract, EE) between 3.95% and 5.39%, the content of acid detergentfiber (ADF) between 24.39% and 39.07%, the content of neutral detergent fiber (NDF)between 29.92% and 41.92%, the content of nitrogen free extract (NFE) between 52.25% and59.99%, the content of calcium between 0.21% and 0.35%, and the content of phosphorusbetween 0.22% and 0.40%. In terms of the content of crude protein and fat, CQ-3 andPS-07 at the flowering stage might have relatively more advantages as forage. Ash in eighttartary buckwheat varieties at the pustulation stage varied between 7.70% and 12.65% CPbetween 3.80% and 5.93%, CF between 9.04% and 12.85%, EE between 3.70% and 4.54%,ADF between 28.14% and 33.07%, NDF between 31.77% and 50.04%, NFE between 56.06%and 65.99%, calcium between 0.21% and 0.32%, and phosphorus between 0.23% and 0.38%(Table 1). The advantage of CQ-3 and PS-07 at the pustulation stage as forage is, however,not obvious in terms of the content of crude protein and fat. Ash in eight tartary buckwheatvarieties of the mature stage varied between 7.38% and 11.76%, CP between 3.44% and6.96%, CF between 7.45% and 20.20%, EE between 3.42% and 5.39%, ADF between 33.20%and 42.02%, NDF between 31.34% and 45.17%, NFE between 51.52% and 68.35%, calciumbetween 0.20% and 0.32%, and the phosphorus between 0.16% and 0.25% (Table 1). Theadvantage of CQ-3 at the mature stage as forage becomes relatively more obvious in termsof the content of fat. Overall, ash of PS-07 at the flowering stage is the highest of 17.49%,CP of CQ-3 at the flowering stage is the highest of 7.08%, CF of YQ-1 at the mature stage isthe highest of 20.20%, EE of PS-07 at the flowering stage is the highest of 5.39%, ADF ofEWPS at the mature stage is the highest of 42.02%, NDF of ZK-1 at the pustulation stage isthe highest of 50.04%, NFE of M-09125 at the mature stage is the highest of 68.35%, calciumcontent of CQ-3 at the flowering stage is the highest of 0.35%, phosphorus content of ZK-1at the flowering stage is the highest of 0.40%.

Int. J. Plant Biol. 2022, 13 36

Table 1. Nutritional contents of eight tartary buckwheat varieties at different growth stages on a dry matter basis (%).

Stages Varieties Ash CP CF EE ADF NDF NFE Calcium Phosphorus

Flowering stage

M-09125 15.69 ± 0.09 ab 5.06 ± 0.14 cd 10.24 ± 0.18 bcd 3.95 ± 0.06 a 26.98 ± 0.26 c 36.46 ± 0.64 cd 56.73 ± 0.18 b 0.29 ± 0.01 cd 0.38 ± 0.00 b

XQ-1 15.39 ± 0.03 ab 3.15 ± 0.09 a 9.99 ± 1.30 bc 4.06 ± 0.08 ab 29.03 ± 0.08 e 41.92 ± 0.83 e 59.99 ± 0.87 c 0.34 ± 0.00 b 0.32 ± 0.00 f

BY 16.30 ± 0.03 ab 3.74 ± 0.13 b 8.70 ± 0.60 b 4.31 ± 0.06 c 39.07 ± 0.90 f 37.16 ± 0.16 d 58.14 ± 0.02 bc 0.21 ± 0.00 a 0.22 ± 0.00 a

EWPS 16.27 ± 0.04 ab 3.95 ± 0.30 b 13.50 ± 1.00 f 5.01 ± 0.10 d 24.72 ± 0.29 a 34.44 ± 0.49 b 53.25 ± 1.40 a 0.34 ± 0.00 c 0.37 ± 0.00 f

ZK-1 16.15 ± 0.13 ab 5.50 ± 0.05 e 12.31 ± 0.20 ef 4.15 ± 0.05 bc 25.59 ± 0.54 b 37.35 ± 0.89 d 54.30 ± 0.10 a 0.31 ± 0.00 e 0.40 ± 0.00 c

PS-07 17.49 ± 0.07 a 4.83 ± 0.20 c 11.70 ± 1.30 de 5.39 ± 0.19 e 24.39 ± 0.33 a 29.92 ± 0.43 a 52.25 ± 1.52 a 0.33 ± 0.00 d 0.39 ± 0.00 e

CQ-3 15.14 ± 3.46 ab 7.08 ± 0.07 f 6.00 ± 0.75 a 4.88 ± 0.07 d 28.10 ± 0.23 d 33.42 ± 0.63 b 57.19 ± 2.70 b 0.35 ± 0.01 cd 0.38 ± 0.00 g

YQ-1 14.54 ± 0.03 b 5.21 ± 0.11 d 10.48 ± 0.96 cd 4.06 ± 0.08 ab 26.62 ± 0.53 c 35.83 ± 0.80 c 57.84 ± 0.58 bc 0.32 ± 0.00 b 0.31 ± 0.00 d

Pustulationstage

M-09125 7.70 ± 0.06 a 5.69 ± 0.15 cd 10.38 ± 0.53 b 3.72 ± 0.02 b 31.76 ± 0. 51 bcd 47.74 ± 0. 66 e 65.36 ± 0.45 e 0.27 ± 0.00 c 0.31 ± 0.00 b

XQ-1 11.91 ± 0.00 c 5.93 ± 0.20 d 9.96 ± 0.39 ab 4.54 ± 0.04 e 30.86 ± 0. 66 bc 39.95 ± 0. 36 b 59.11 ± 0.25 b 0.27 ± 0.00 e 0.34 ± 0.00 b

BY 12.65 ± 0.10 d 5.53 ± 0.22 c 12.85 ± 0.85 d 4.00 ± 0.06 cd 30.51 ± 0. 20 b 31.77 ± 0. 31 c 56.06 ± 0.16 a 0.27 ± 0.00 d 0.32 ± 0.00 b

EWPS 9.64 ± 0.06 c 4.52 ± 0.16 b 10.84 ± 0.52 bc 3.70 ± 0.07 b 28.14 ± 0.05 a 41.82 ± 0.55 a 63.91 ± 0.47 d 0.30 ± 0.00 b 0.29 ± 0.00 c

ZK-1 9.84 ± 0.28 c 4.30 ± 0.11 b 11.68 ± 0.91 cd 3.70 ± 0.06 b 28.89 ± 0. 87 a 50.04 ± 0.74 f 63.10 ± 0.45 cd 0.30 ± 0.00 g 0.38 ± 0.00 c

PS-07 8.94 ± 0.02 b 3.82 ± 0.08 a 11.15 ± 1.04 bc 3.39 ± 0.07 a 33.03 ± 1.63 d 44.01 ± 0.39 d 64.92 ± 0.84 e 0.32 ± 0.00 g 0.37 ± 0.00 d

CQ-3 9.12 ± 0.03 b 3.80 ± 0.20 a 9.04 ± 0.62 a 3.91 ± 0.00 c 33.07 ± 0.20 d 48.42 ± 0.29 e 65.99 ± 0.70 f 0.21 ± 0.00 f 0.35 ± 0.00 a

YQ-1 8.96 ± 0.10 b 3.95 ± 0.11 a 12.00 ± 0.27 cd 4.02 ± 0.04 d 32.12 ± 0.23 cd 42.07 ± 1.13 c 62.74 ± 0.09 c 0.27 ± 0.00 a 0.23 ± 0.00 b

Mature stage

M-09125 7.38 ± 0.03 a 4.73 ± 0.12 c 8.60 ± 1.07 a 4.19 ± 0.10 c 36.57 ± 0.53 c 39.77 ± 0.84 ab 68.35 ± 0.50 f 0.23 ± 0.00 c 0.16 ± 0.00 a

XQ-1 8.11 ± 0.07 c 6.96 ± 0.10 f 11.07 ± 1.97 b 3.76 ± 0.07 b 39.07 ± 0.90 d 44.36 ± 0.72 b 61.52 ± 0.23 c 0.22 ± 0.00 b 0.21 ± 0.00 c

BY 10.38 ± 0.04 f 5.10 ± 0.06 d 16.77 ± 0.75 c 3.42 ± 0.20 a 33.20 ± 0.32 a 45.17 ± 0.57 b 56.95 ± 0.01 a 0.22 ± 0.01 b 0.21 ± 0.00 c

EWPS 9.16 ± 0.01 d 5.79 ± 0.17 e 12.43 ± 1.11 b 4.16 ± 0.08 c 42.02 ± 0.23 e 42.50 ± 0.16 b 59.92 ± 0.03 b 0.30 ± 0.00 e 0.22 ± 0.01 d

ZK-1 9.89 ± 0.14 e 5.15 ± 0.08 d 11.74 ± 0.34 b 3.88 ± 0.10 b 38.99 ± 0.02 d 43.56 ± 0.97 b 61.72 ± 0.25 c 0.28 ± 0.00 d 0.23 ± 0.00 e

PS-07 11.76 ± 0.54 g 5.36 ± 0.22 d 7.45 ± 1.05 a 4.72 ± 0.05 d 35.79 ± 0.45 b 31.34 ± 1.21 a 64.18 ± 0.01 d 0.32 ± 0.01 f 0.25 ± 0.00 f

CQ-3 7.77 ± 0.09 bc 4.08 ± 0.39 b 8.71 ± 0.67 a 5.23 ± 0.03 e 35.61 ± 0.35 b 41.88 ± 0.29 b 66.31 ± 0.51 e 0.30 ± 0.00 e 0.25 ± 0.00 f

YQ-1 7.66 ± 0.13 ab 3.44 ± 0.07 a 20.20 ± 2.21 d 3.79 ± 0.09 b 33.70 ± 0.20 a 38.84 ± 0.77 ab 56.55 ± 1.72 a 0.20 ± 0.00 a 0.18 ± 0.00 b

M-09125: Meng-09125; XQ-1: Xiqiao No. 1; BY: Buyue; EWPS: Ewu Pedgree selection; ZK-1: Zhaoku No. 1; PS-07: Pedgree selection-07; CQ-3: Chuanqiao No.3 and YQ-1: Youqiao No. 1.The data in the table are represented as the mean ± standard deviation. Different lowercase letters on each column indicate significant differences between stages at p < 0.05.

Int. J. Plant Biol. 2022, 13 37

3.2. Total Flavonoids and Rutin Contents of Eight Tartary Buckwheat Varieties

Results showed that CQ-3 has the highest total flavonoids content at the floweringstage, while M-09125 and XQ-1 have the highest total flavonoids content at the pustulationstage and mature stage, respectively (Figure 1). Similarly, CQ-3 has the highest rutin contentat the flowering stage, while M-09125 and CQ-3 have the highest content of rutin at thepostulation and mature stage, respectively (Figure 2). In general, our results indicated thatthe content of total flavonoids of tartary buckwheat varieties M-09125 and XQ-1 were thehighest at the pustulation stage and at the mature stage, respectively, and rutin content oftartary buckwheat varieties M-09125 and XQ-1 were the highest at the pustulation stageand at the flowering stage, respectively, while the total flavonoids and rutin contents of allother six tartary buckwheat varieties were the highest at the flowering stage.

Figure 1. Total flavonoids content of tartary buckwheat varieties at different growth stages. Thevalue is flavonoids (mg) per gram dry matter basis. M-09125: Meng-09125; XQ-1: Xiqiao No. 1;BY: Buyue; EWPS: Ewu Pedgree selection; ZK-1: Zhaoku No. 1; PS-07: Pedgree selection-07; CQ-3:Chuanqiao No.3 and YQ-1: Youqiao No. 1. Different lowercase letters on each column indicatesignificant differences between stages at p < 0.05.

Figure 2. Rutin content of tartary buckwheat varieties at different growth stages. The value is rutin(mg) per gram dry matter basis. M-09125: Meng-09125; XQ-1: Xiqiao No. 1; BY: Buyue; EWPS: EwuPedgree selection; ZK-1: Zhaoku No. 1; PS-07: Pedgree selection-07; CQ-3: Chuanqiao No. 3 andYQ-1: Youqiao No. 1. Different lowercase letters on each column indicate significant differencesbetween stages at p < 0.05.

3.3. Relative Feed Value and Relative Forage Quality Analysis of Eight Tartary BuckwheatVarieties

Based on the data in Table 1, the dry matter intakes (DMI), digestible dry matter(DDM), and total digestible nutrients (TDN) were calculated (Table 2), with which therelative feed value (RFV) and relative forage quality (RFQ) were also calculated (Table 2).Results showed that all these eight tartary buckwheat varieties at three growth stagespresented high index values of RFV (121.31% to 217.39%) and RFQ (117.26% to 224.54%),indicating that they could be considered as forage candidates with high quality. However,

Int. J. Plant Biol. 2022, 13 38

based on the RFV and RFQ values, BY might be more suitable to be popularized for forageharvesting at the pustulation stage, while the other seven tartary buckwheat varieties mightbe more suitable to be popularized for forage harvesting at the flowering stage.

Table 2. The dry matter intakes, digestible dry matter, total digestible nutrients, relative feed valueand relative forage quality of eight tartary buckwheat varieties at different growth stages.

Stages Varieties DMI(%DM) DDM(%DM) TDN(%DM) RFV RFQ

Flowering stage

M-09125 3.69 ± 0.66 bc 67.88 ± 0.20 d 62.12 ± 0.19 d 173.23 ± 3.51 b 186.29 ± 32.64 b

XQ-1 3.29 ± 0.73 a 66.29 ± 0.06 b 60.58 ± 0.06 b 147.12 ± 2.82 a 162.20 ± 35.79 a

BY 3.18 ± 0.09 b 58.47 ± 0.70 a 53.04 ± 0.68 a 146.36 ± 1.30 a 137.16 ± 5.69 a

EWPS 3.93 ± 0.80 d 69.64 ± 0.22 f 63.82 ± 0.22 f 188.13 ± 3.16 c 204.03 ± 42.21 c

ZK-1 3.73 ± 0.83 b 68.96 ± 0.42 e 63.16 ± 0.40 e 171.81 ± 4.39 b 191.89 ± 43.66 b

PS-07 4.31 ± 0.53 f 69.90 ± 0.26 f 64.07 ± 0.25 f 217.39 ± 3.89 d 224.54 ± 26.76 d

CQ-3 3.83 ± 0.39 e 67.01 ± 0.18 c 61.28 ± 0.17 c 186.59 ± 3.08 c 190.74 ± 19.89 c

YQ-1 3.71 ± 0.69 c 68.16 ± 0.41 d 62.39 ± 0.40 d 176.99 ± 3.45 b 188.16 ± 33.88 b

Pustulationstage

M-09125 2.94 ± 0.73 b 64.16 ± 0.51 abc 58.53 ± 0.50 abc 125.03 ± 0.57 a 140.05 ± 35.77 a

XQ-1 3.31 ± 0.50 e 64.68 ± 0.51 bc 59.21 ± 0.49 bc 151.03 ± 1.79 d 159.17 ± 22.94 c

BY 3.82 ± 0.10 f 65.13 ± 0.15 c 59.47 ± 0.15 c 190.72 ± 1.66 e 184.56 ± 5.38 d

EWPS 3.34 ± 0.80 d 66.98 ± 0.04 d 61.24 ± 0.04 d 149.01 ± 1.86 d 166.11 ± 40.17 c

ZK-1 2.97 ± 1.03 a 66.39 ± 0.68 d 60.68 ± 0.66 d 123.43 ± 2.15 a 146.81 ± 52.23 a

PS-07 3.04 ± 0.52 c 63.17 ± 1.27 a 57.57 ± 1.22 a 133.53 ± 2.78 b 142.51 ± 27.03 b

CQ-3 2.86 ± 0.67 b 63.14 ± 0.16 a 57.55 ± 0.15 a 121.31 ± 0.70 a 133.69 ± 31.64 a

YQ-1 3.15 ± 0.52 d 63.88 ± 0.18 ab 58.26 ± 0.17 ab 141.32 ± 4.17 c 149.06 ± 24.87 c

Mature stage

M-09125 3.23 ± 0.05 d 60.41 ± 0.41 c 54.92 ± 0.40 c 147.98 ± 3.67 d 144.04 ± 3.07 d

XQ-1 2.84 ± 0.20 ab 58.47 ± 0.70 b 53.04 ± 0.68 b 122.65 ± 3.27 a 122.35 ± 7.33 a

BY 2.96 ± 0.56 c 63.04 ± 0.25 e 57.45 ± 0.24 e 129.82 ± 1.62 b 137.16 ± 5.69 b

EWPS 2.84 ± 0.02 bc 56.17 ± 0.18 a 50.83 ± 0.17 a 122.94 ± 0.67 a 138.47 ± 26.91 a

ZK-1 2.88 ± 0.17 ab 58.52 ± 0.02 b 53.10 ± 0.02 b 125.01 ± 2.76 ab 117.26 ± 0.31 ab

PS-07 3.71 ± 0.33 e 61.02 ± 0.35 d 55.50 ± 0.34 d 181.28 ± 6.31 e 124.50 ± 7.36 e

CQ-3 3.04 ± 0.29 c 61.16 ± 0.27 d 55.64 ± 0.26 d 135.86 ± 1.46 c 167.28 ± 14.33 c

YQ-1 3.22 ± 0.29 d 62.65 ± 0.16 e 57.07 ± 0.15 e 150.10 ± 2.99 d 137.49 ± 13.56 d

M-09125: Meng-09125; XQ-1: Xiqiao No. 1; BY: Buyue; EWPS: Ewu Pedgree selection; ZK-1: Zhaoku No. 1; PS-07:Pedgree selection-07; CQ-3: Chuanqiao No.3 and YQ-1: Youqiao No. 1. The data in the table are representedas the mean ± standard deviation. Different lowercase letters on each column indicate significant differencesbetween stages at p < 0.05.

3.4. Principal Component Analysis

With the assayed contents of CP, CF, EE, ADF, NDF, NFE, calcium, phosphorus,total flavonoids, and rutin, principal components analysis (PCA) was performed. Resultsshowed that two components (PC1 and PC2) retained from PCA at the flowering stage,explaining 68.888% of total variability of original data (Table S1), indicating PC1 and PC2mainly represent most of the data contained in original indices, while principal componentswere retained from the pustulation (Table S2) and mature stages (Table S3), explaining54.025% and 66.319% of total variability of original data, respectively. The eigenvectormatrix reflects the load of each index on each principal component. Data in Table S1showed that in PC1, EE, NDF, and NFE, calcium and ADF had higher loads and werethe main factors of this principal component, while, in PC2, rutin and total flavonoidswere the main factors and had relatively large loads. Thus, PC1 mainly represented thepossibility of metabolic and energy supply, digestibility, and the mineral element contentof tartary buckwheat varieties, while PC2 mainly represented the resistance to oxidationof tartary buckwheat varieties. In Table S2, while the tartary buckwheat varieties at thepustulation stage, NFE and NDF had higher loads and were the main factors of PC1, whichmainly represented the possibility of metabolic of tartary buckwheat varieties, while rutin,total flavonoids, and EE were the main factors of PC2, which represented the resistance tooxidation. Results in Table S3 showed that calcium, calcium, CF, EE, total flavonoids, and

Int. J. Plant Biol. 2022, 13 39

NFE had higher loads and were the main factors of PC1 at the mature stage of the tartarybuckwheat varieties, which mainly represented the mineral element content, the possibilityof metabolic and energy supply, and the resistance to oxidation of tartary buckwheatvarieties, while CP and ADF were the main factors of PC2, which represented proteinnutritive value and digestibility of tartary buckwheat varieties. In summary, these indicescould be used as comprehensive indicators for evaluating the quality of tartary buckwheatvarieties as forage. In addition, the single score of one principal component was calculatedas F, and then the comprehensive score of different tartary buckwheat varieties was furthercalculated as Dn based on the F values (Tables S4–S6). Results showed that PS-07 obtainedthe highest score among the eight tartary buckwheat varieties at the flowering stage, whichwas consistent with the result of RFQ and RFV analysis.

4. Discussion

Consumers are paying more and more attention to the quality of livestock products,and thus quality and safety of forage as well [31]. At present, commonly used foragescan meet the basic nutritional requirements in the process of livestock feeding, but addi-tional additives are usually needed for comprehensive nutrition, especially to improvethe quality of meat and milk, improve the litter rate, and enhance immune and diseaseresistance [18–20]. The whole plant of tartary buckwheat as a promising feeding crop,including seeds, flowers, leaves, and stems, is rich in bioactive components, which playan important and complex physiological regulation role in tartary buckwheat forage-fedlivestock [32]. Here, the major nine forage nutrient index were analyzed (including Ash,CP, CF, EE, ADF, NDF, NFE, calcium, and phosphorus content) of the whole plant of eighttartary buckwheat varieties, and the rutin content and total flavonoids as well to exploretheir potential forage values and quality. Our data showed that CQ-3 at the floweringstage contained the highest content of CP, followed by XQ-1 at pustulation and maturestages. PS-07 at the flowering stage contained the lowest content of NDF, followed by BY atthe pustulation stage and CQ-3 at the flowering stage. PS-07 at the flowering stage alsocontained the lowest level of ADF, but followed by EWPS and ZK-1 at the flowering stages.It is known that more attention is usually paid to the content of CP, ADF, and NDF inview of the forage nutritional index. It was reported that NDF and ADF were related tothe forage DM digestibility of ruminant animals, and the CP content was regarded as thedecisive factor in the nutritional value of forage for herbivores [33]. Meanwhile, the othersix nutritional content indices of these eight tartary buckwheat varieties all met the stan-dard range of forage. Taking the assayed nutritional contents (Table 1) into comprehensiveconsideration, CQ-3 and PS-07 at the flowering stage are thus suggested to be relativelymore suitable for harvesting as forage, although all the eight tartary buckwheat varietiesare suitable for forage.

In order to exactly evaluate the potential forage value for these eight tartary buckwheatvarieties, RFV and RFQ analysis were further performed. According to the definition ofRFV value, if the value is higher than 100, it indicates that the nutritional value of theplant met the requirements for forage selection [34]. Moreover, the higher RFV means thehigher potential nutritional value of forage crop [35]. Turk et al. concluded that pea grassharvested in the early growth stage with high nutritional value displayed high RFV of212.5 [36]. Gürsoy et al. showed that brogated varigatus had high RFQ and thus is widelyused to eliminate the quality roughage deficit [37]. Our data showed that, at the floweringstage, PS-07 displayed the highest level of RFV and RFQ, indicating that BY at the floweringstage is the most relatively suitable for forage and feed application. At the pustulationstage, however, BY displayed the highest level of RFV and RFQ, indicating that PS-07 atthe pustulation stage is the most relatively suitable for forage and feed application. Atthe mature stage, PS-07 has the highest level of RFV, while CQ-3 has the highest level ofRFQ, suggesting that PS-07 at the mature stage is the most relatively suitable for forageand CQ-3 at the mature stage most suitable for feed application. Taking RFV and RFQ in

Int. J. Plant Biol. 2022, 13 40

consideration together, PS-07, BY, and CQ-3 are relatively more suitable for forage, whichis consistent with the above findings as a whole.

In addition, PCA was performed for these eight tartary buckwheat varieties. PCA wasapplied to simplify the interpretation of results and narrow down the number of variablesthat are mainly related to specific parameters of interest. PCA ranked the samples with thefactors that eigenvalue is more than one to reduce errors as far as possible, which couldbe seemed as a comprehensive evaluation of forage crop [38]. As it is shown in Table S1,when harvested at the flowering stage for forage, CQ-3 might be ranked at the Top 1 basedon PCA, followed by PS-07. When harvested at the mature stage for forage, CQ-3 mightstill be ranked at Top 1, followed by XQ-1 and PS-07 (Table S6). Although the PCA rankingwhen harvested at the pustulation stage used as forage is not exactly the same as thatharvested at the flowering stage and mature stage, CQ-3 and PS-07 still are considered tobe more relatively suitable for forage under comprehensive comparison, which is generallyin agreement with that from RFV and RFQ analysis, as well as the analysis of nine majornutrient indexes.

Buckwheat is regarded as a crop possessing high nutritional value [39], and especiallyis the only source of dietary rutin among the cereals and pseudocereals’ crops [40]. It isknown that rutin is the main content of tartary buckwheat flavonoid. Tartary buckwheatrutin is not only a potential drug material with high medicinal value to humans [41], butalso has lots of beneficial effects to animals in the field of forage. Hassan et al. found that thesupplemented broiler daily ration with rutin could promote its growth and suppress lipo-genesis [42]. Leiber et al. verified that buckwheat rutin could improve rumen fermentationof dairy cows [43]. In addition, Sayed et al. reported that feeding 20% or more buckwheatcould have a positive effect on the lipid profiles of broilers [44]. Other researchers alsoreported that tartary buckwheat protein can help to lower cholesterol [45,46]. Therefore,the rutin content and total flavonoids were determined in the whole plant of these eighttartary buckwheat varieties at different stages. Our data showed that the whole plant ofthe eight tartary buckwheat varieties were rich in flavonoids at all three growth stages, andthe content of total flavonoids in tartary buckwheat varieties PS-07 and CQ-3 reached thehighest at the flowering stage. Accordingly, the whole plant of the eight tartary buckwheatvarieties were rich in rutin, and the PS-07 and CQ-3 at the flowering stage also containedthe highest rutin content. Based on the comprehensive analysis, CQ-3 and PS-07 harvestedat the flowering stage when used as forage might more effectively improve the function offorage to promote livestock immunity and disease resistance.

5. Conclusions

In this study, we identified that eight tartary buckwheat varieties are suitable for forageapplication, among which tartary buckwheat varieties PS-07 and CQ-3 have relativelyhigher potential forage value especially when harvested at the flowering stage. Ourresearch will greatly benefit the forage use of these eight tartary buckwheat varieties andprovide a theoretical basis for the best harvest stage of these tartary buckwheat varieties asforage.

Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijpb13020005/s1, Table S1: Principal component eigenvector,eigenvalue, contribution rate and accumulative contribution rate of eight tartary buckwheat varietiesat flowering stage, Table S2: Principal component eigenvector, eigenvalue, contribution rate andaccumulative contribution rate of eight tartary buckwheat varieties at pustulation stage, Table S3:Principal component eigenvector, eigenvalue, contribution rate and accumulative contribution rateof eight tartary buckwheat varieties at mature stage, Table S4: Comprehensive scoring of principalcomponent values of eight tartary buckwheat varieties at flowering stage, Table S5: Comprehensivescoring of principal component values of eight tartary buckwheat varieties at pustulation stage, TableS6: Comprehensive scoring of principal component values of eight tartary buckwheat varieties atmature stage.

Int. J. Plant Biol. 2022, 13 41

Author Contributions: Data curation, M.Z. (Mengjie Zhou), M.H., and J.W. (Jiankang Wang); Investi-gation, J.W. (Junzhen Wang); Methodology, T.S.; Writing—original draft, M.Z. (Mengjie Zhou) andZ.L.; Writing—review and editing, M.Z. (Meiliang Zhou), F.L. and Z.L. All authors have read andagreed to the published version of the manuscript.

Funding: This work was supported by the National Key R & D Program of China (2017YFE0117600),the Key Research and Development Projects of Zhejiang Province (2022C04003).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The data presented in this study are available on request from thecorresponding author.

Acknowledgments: We acknowledge the College of Life and Environmental Science, WenzhouUniversity, Wenzhou, China, for providing the laboratory facilities, and thank Mengqi Qing forhelping us to modify the manuscript.

Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

Abbreviation Full NameXQ-1 Xiqiao No. 1YQ-1 Youqiao No. 1M-09125 Meng-09125CQ-3 Chuanqiao No. 3ZK-1 Zhaoku No. 1EWPS Ewu pedgree selectionBY BuyuePS-07 Pedgree selection-07CP Crude proteinCF Crude fiberEE Crude fat contentADF Acid detergent fiberNDF Neutral detergent fiberNFE Nitrogen free extractRFV Relative feed valueRFQ Relative forage qualityDMI Dry matter intakeDDM Digestible dry matterTDN Total digestible nutrients

References1. Yao, H.; Li, C.; Zhao, H.; Zhao, J.; Chen, H.; Bu, T.; Anhu, W.; Wu, Q. Deep sequencing of the transcriptome reveals distinct

flavonoid metabolism features of black tartary buckwheat (Fagopyrum tataricum Garetn.). Prog. Biophys. Mol. Biol. 2017, 124, 49–60.[CrossRef] [PubMed]

2. Singh, M.; Malhotra, N.; Sharma, K. Buckwheat (Fagopyrumsp.) genetic resources: What can they contribute towards nutritionalsecurity of changing world? Genet. Resour. Crop Evol. 2020, 67, 1639–1658. [CrossRef]

3. Aubert, L.; Konradova, D.; Barris, S.; Quinet, M. Different drought resistance mechanisms between two buckwheat speciesFagopyrum esculentum and Fagopyrum tataricum. Physiol. Plant. 2021, 172, 577–586. [CrossRef] [PubMed]

4. Lu, Q.H.; Wang, Y.Q.; Yang, H.B. Effect of exogenous calcium on physiological characteristics of salt tolerance in tartary buckwheat.Biologia 2021, 76, 3621–3630. [CrossRef]

5. Er, M.; Keles, G. Buckwheat conservation as hay or silage: Agronomic evaluation, nutritive value, conservation quality, andintake by lactating dairy goats. Trop Anim. Health Prod. 2021, 53, 215–222. [CrossRef]

6. Liu, N.; Zeller, F.J.; Chen, Q.F. The flavonoid content in leaves and inflorescences of the wild perennial Fagopyrum cymosumcomplex. Genet. Resour. Crop Evol. 2013, 60, 825–838. [CrossRef]

7. Kalac, P.; Samkova, E. The effects of feeding various forages on fatty acid composition of bovine milk fat: A review. Czech J. Anim.Sci. 2010, 55, 521–537. [CrossRef]

Int. J. Plant Biol. 2022, 13 42

8. Eugene, M.; Klumpp, K.; Sauvant, D. Methane mitigating options with forages fed to ruminants. Grass Forage Sci. 2021, 76,196–204. [CrossRef]

9. Song, Y.; Lv, J.; Ma, Z.; Dong, W. The mechanism of alfalfa (Medicago Sativa L.) response to abiotic stress. Plant Growth Regul. 2019,89, 239–249. [CrossRef]

10. Jonker, A.; Yu, P. The role of proanthocyanidins complex in structure and nutrition interaction in alfalfa forage. Int. J. Mol. Sci.2016, 17, 793. [CrossRef]

11. Silva, T.N.; Thomas, J.B.; Dahlberg, J.; Rhee, S.Y.; Mortimer, J.C. Progress and challenges in sorghum biotechnology, a multipurposefeedstock for the bioeconomy. J. Exp. Bot. 2022, 73, 646–664. [CrossRef] [PubMed]

12. Klopfenstein, T.J.; Erickson, G.E.; Berger, L.L. Maize is a critically important source of food, feed, energy and forage in the USA.Field Crop. Res. 2013, 153, 5–11. [CrossRef]

13. Jonker, A.; Yu, P. The Occurrence, Biosynthesis, and molecular structure of proanthocyanidins and their effects on legume forageprotein precipitation, digestion and absorption in the ruminant digestive tract. Int. J. Mol. Sci. 2017, 18, 1105. [CrossRef] [PubMed]

14. Balota, M.; Payne, W.A.; Veeragom, S.K.; Stewart, B.A.; Rosenow, D.T. Respiration and its relationship to germination, emergence,and early growth under cool temperatures in sorghum. Crop Sci. 2010, 50, 1414–1422. [CrossRef]

15. Huang, Z.; Dunkerley, D.; Lopez-Vicente, M.; Wu, G.L. Trade-offs of dryland forage production and soil water consumption in asemi-arid area. Agric. Water Manag. 2020, 241, 106349. [CrossRef]

16. Huang, Q.; Liu, X.; Zhao, G.; Hu, T.; Wang, Y. Potential and challenges of tannins as an alternative to in-feed antibiotics for farmanimal production. Anim. Nutr. 2018, 4, 137–150. [CrossRef]

17. Chattopadhyay, M.K. Use of antibiotics as feed additives: A burning question. Front. Microbiol. 2014, 5, 334. [CrossRef]18. Sedej, I.; Sakac, M.; Mandic, A.; Misan, A.; Tumbas, V.; Canadanovic-Brunet, J. Buckwheat (Fagopyrum esculentum moench) grain

and fractions: Antioxidant compounds and activities. J. Food Sci. 2012, 77, C954–C959. [CrossRef]19. Idowu, A.T.; Olatunde, O.O.; Adekoya, A.E.; Idowu, S. Germination: An alternative source to promote phytonutrients in edible

seeds. Food Qual. Saf. 2020, 4, 129–133. [CrossRef]20. Luthar, Z.; Golob, A.; Germ, M.; Vombergar, B.; Kreft, I. Tartary buckwheat in human nutrition. Plants 2021, 10, 700. [CrossRef]21. Cui, K.; Wang, Q.; Wang, S.; Diao, Q.; Zhang, N. The facilitating effect of tartary buckwheat flavonoids and lactobacillus plantarum

on the growth performance, nutrient digestibility, antioxidant capacity, and fecal microbiota of weaned piglets. Animals 2019, 9,986. [CrossRef] [PubMed]

22. Scuderi, R.A.; Lam, Y.W.; Ebenstein, D.B.; Tacoma, R.; Cersosimo, L.M.; Kraft, J.; Brito, A.F.; Greenwood, S.L. Comparativeanalysis of the skim milk and milk fat globule membrane proteomes produced by jersey cows grazing pastures with differentplant species diversity. J. Dairy Sci. 2020, 103, 7498–7508. [CrossRef] [PubMed]

23. Amelchanka, S.L.; Kreuzer, M.; Leiber, F. Utility of buckwheat (Fagopyrum esculentum moench) as feed: Effects of forage and grainon in vitro ruminal fermentation and performance of dairy cows. Anim. Feed Sci. Technol. 2010, 155, 111–121. [CrossRef]

24. Neu, A.E.; Sheaffer, C.C.; Undersander, D.J.; Hall, M.H.; Kniffen, D.M.; Wells, M.S.; Catalano, D.N.; Martinson, K.L. Hay rake-typeeffect on ash and forage nutritive values of alfalfa hay. Agron. J. 2017, 109, 2163–2171. [CrossRef]

25. D’Heer, B.; Boever, J.; Vanacker, J.M.; Boucqué, C.V. The filter bag versus the conventional filtration technique for the determinationof crude fibre and Van Soest cell wall constituents. J. Anim. Feed Sci. 2000, 9, 513–526. [CrossRef]

26. Thiex, N.J.; Anderson, S.; Gildemeister, B. Crude fat, diethyl ether extraction, in feed, cereal grain, and forage (ran-dall/soxtec/submersion method): Collaborative study. J. Aoac. Int. 2003, 86, 888–898. [CrossRef]

27. Prantil, L.R.; Heinze, C.R.; Freeman, L.M. Comparison of carbohydrate content between grain-containing and grain-free dry catdiets and between reported and calculated carbohydrate values. J. Feline Med. Surg. 2018, 20, 349–355. [CrossRef]

28. Bhandari, S.; Khadayat, K.; Poudel, S.; Shrestha, S.; Shrestha, R.; Devkota, P.; Khanal, S.; Marasini, B.P. Phytochemical analysis ofmedicinal plants of nepal and their antibacterial and antibiofilm activities against uropathogenic Escherichia coli. BMC Complement.Med. Ther. 2021, 21, 116. [CrossRef]

29. Kim, J.; Jho, K.H.; Choi, Y.H.; Nam, S.Y. Chemopreventive effect of cactus (opuntia humifusa) extracts: Radical scavenging activity,pro-apoptosis, and anti-Inflammatory effect in human colon (SW480) and breast cancer (MCF7) cells. Food Funct. 2013, 4, 681–688.[CrossRef]

30. Ward, R.; de Ondarza, M.B. Relative Feed Value (RFV) vs. Relative Forage Quality (RFQ). 6. Available online: https://www.foragelab.com/Media/RFV_vs_RFQ-CVASPerspective.pdf (accessed on 14 February 2008).

31. Singer, S.D.; Weselake, R.J.; Acharya, S. Molecular enhancement of alfalfa: Improving quality traits for superior livestockperformance and reduced environmental impact. Crop Sci. 2018, 58, 55–71. [CrossRef]

32. Li, J.; Yang, P.; Yang, Q.; Gong, X.; Ma, H.; Dang, K.; Chen, G.; Gao, X.; Feng, B. Analysis of flavonoid metabolites in buckwheatleaves using UPLC-ESI-MS/MS. Molecules 2019, 24, 1310. [CrossRef] [PubMed]

33. Lemaire, G.; Belanger, G. Allometries in plants as drivers of forage nutritive value: A review. Agriculture 2019, 10, 5. [CrossRef]34. Rohweder, D.A.; Barnes, R.; Jorgensen, N. Proposed hay grading standards based on laboratory analyses for evaluating quality. J.

Anim. Sci. 1978, 47, 747–759. [CrossRef]35. Geng, Y.; Ranjitkar, S.; Yan, Q.; He, Z.; Su, B.; Gao, S.; Niu, J.; Bu, D.; Xu, J. Nutrient value of wild fodder species and the

implications for improving the diet of mithun (Bos Frontalis) in Dulongjiang area, Yunnan province, China. Plant Divers. 2020, 42,455–463. [CrossRef] [PubMed]

Int. J. Plant Biol. 2022, 13 43

36. Turk, M.; Albayrak, S. Effect of harvesting stages on forage yield and quality of different leaf types pea cultivar. Turk. J. FieldCrops 2012, 17, 111–114.

37. Gürsoy, E.; Kaya, A.; Gül, M. Determining the nutrient content, energy, and in vitro true digestibility of some grass forage plants.Emir. J. Food Agric. 2021, 33, 417–422. [CrossRef]

38. Gallo, A.; Moschini, M.; Cerioli, C.; Masoero, F. Use of principal component analysis to classify forages and predict their calculatedenergy content. Animal 2013, 7, 930–939. [CrossRef]

39. Maxin, G.; Graulet, B.; Le Morvan, A.; Picard, F.; Portelli, J.; Andueza, D. Cover crops as alternative forages for ruminants:Nutritive characteristics, in vitro digestibility, methane and ammonia production. Anim. Prod. Sci. 2020, 60, 823–832. [CrossRef]

40. Lu, L.; Murphy, K.; Baik, B.K. Genotypic variation in nutritional composition of buckwheat groats and husks. Cereal Chem. 2013,90, 132–137. [CrossRef]

41. Ruan, J.J.; Zhou, Y.X.; Yan, J.; Zhou, M.L.; Woo, S.H.; Weng, W.F.; Cheng, J.P.; Zhang, K.X. Tartary Buckwheat: An Under-utilizededible and medicinal herb for food and nutritional security. Food Res. Int. 2020. [CrossRef]

42. Hassan, F.; Roushdy, E.M.; Kishawy, A.; Zaglool, A.W.; Tukur, H.A.; Saadeldin, I.M. Growth performance, antioxidant capacity,lipid-related transcript expression and the economics of broiler chickens fed different levels of rutin. Animals 2019, 9, 7. [CrossRef][PubMed]

43. Leiber, F.; Kunz, C.; Kreuzer, M. Influence of different morphological parts of buckwheat (Fagopyrum esculentum) and its majorsecondary metabolite rutin on rumen fermentation in vitro. Czech J. Anim. Sci. 2012, 57, 10–18. [CrossRef]

44. Sayed, M.; Islam, M.; Haque, M.; Shah, M.; Ahmed, R.; Siddiqui, M.; Hossain, M. Dietary effects of chitosan and buckwheat(Fagopyrum esculentum) on the performance and serum lipid profile of broiler chicks. SA J. Anim. Sci. 2015, 45, 429–440. [CrossRef]

45. Zhang, C.; Zhang, R.; Li, Y.M.; Liang, N.; Zhao, Y.; Zhu, H.; He, Z.; Liu, J.; Hao, W.; Jiao, R.; et al. Cholesterol-lowering activity oftartary buckwheat protein. J. Agric. Food Chem. 2017, 65, 1900–1906. [CrossRef]

46. Liu, J.; Song, Y.; Zhao, Q.; Wang, Y.; Li, C.; Zou, L.; Hu, Y. Effects of tartary buckwheat protein on gut microbiome and plasmametabolite in rats with high-fat diet. Foods 2021, 10, 2457. [CrossRef]